Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition

Abstract

Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits.

Figure 1. NRG1 and erbB4 promote interneuronal dendritic growth

(a) ErbB4 is expressed in mature multipolar cultured cortical interneurons. Scale bar=35uM. (b) Representative traces of DIV28 cortical multipolar interneuron dendritic trees. Axons were omitted for clarity. Neurons expressed either GFP or GFP-tagged kalirin RNAi for 3-days. 1-day posttransfection, neurons were treated with either NRG1β (5nM) or with vehicle for 2 days. Scale bar=100um. (c) Scatter plots showing that NRG1β significantly increases interneuronal dendritic length relative to control neurons treated with vehicle, while the knockdown of kalirin blocks the effect of NRG1β on dendrite length. Knockdown of kalirin by itself did not alter dendrite length. Each point represents the total dendritic length of a single cell. Significance determined using a one-way ANOVA with Tukey post-hoc. Black lines are the mean, n=27–43 cells per condition; *p<0.05 (d) Significant interaction of NRG1β treatment by kalirin expression with regard to total interneuron dendritic length. Significance determined using a 2×2 ANOVA and the interaction term determined. Data are the mean±SEM; *p<0.05 (e) Representative traces of mature cultured interneurons derived from wildtype (WT) mice or from erbB2/B4 conditional knockout (KO) mice. Axons were omitted for clarity. Scale bar=50um. (f) Scatter plots showing that multipolar cultured interneurons from erbB2/B4 KO mice show a reduced total dendritic length relative to WT mice. Each point represents the total dendritic length of a single cell. Significance determined using an unpaired t-test. Black lines are the mean, n=10 cells per condition; *p<0.05 (g) Relative change in total dendritic length of erbB2/B4 KO interneurons as compared to WT interneurons. Significance determined using an unpaired t-test. Data are the mean±SEM; *p<0.05

Figure 2. Kalirin-7 and erbB4 interact and fyn phospho-activates kalirin-7

(a) DIV28 cortical cultures were immunostained for endogenous erbB4 and endogenous kalirin-7. Immunostaining shows a high degree of colocalization between kalirin-7 and erbB4 in interneurons. Scale bar=35uM. (b) 71±4.6% of erbb4 puncta in mature interneuronal dendrites colocalized with kalirin-7. (c) Kalirin-7 was immunoprecipitated from mature cortical cultures and the immunoprecipitate probed for erbB4. Myc was used as an immunoprecipitation control. Kalirin-7 interacted with erbB4. (d) Kalirin-7 was immunoprecipitated from mature cortical cultures and the immunoprecipitate probed with a phospho-tyrosine antibody. Whereas NRG1β (5nM, 3-days) treatment increased kalirin-7 tyrosine phosphorylation, co-treatment with PP2 (10uM) blocked this effect. Graph displays change relative to the vehicle condition and significance determined using a one-way ANOVA with Tukey post-hoc. Data are the mean±SEM, n=3 independent experiments; *p<0.05 (e) hEK293 cells overexpressing various kinases in combination with kalirin-7. Kalirin-7 was immunoprecipitated and the immunoprecipitate probed with a phospho-tyrosine antibody. Of the tested tyrosine kinases, only fyn phosphorylated kalirin-7. (f) hEK293 cells overexpressing fyn and kalirin-7 individually or together, and active Rac1 levels assessed. Fyn in combination with kalirin-7 increased active Rac1 levels beyond that of kalirin-7 or fyn alone. Graph displays change relative to the control condition and significance determined using a one-way ANOVA with Tukey post-hoc. Data are the mean±SEM, n=3 independent experiments; *p<0.05

Figure 3. Fyn increases interneuronal dendritic growth through kalirin, and fyn phosphorylates kalirin-7 through its C-terminus

(a) Representative traces of DIV28 cultured cortical multipolar interneuron dendritic trees. Axons were omitted for clarity. Neurons were transfected with either GFP, GFP-tagged kalirin RNAi, fyn, or GFP-tagged kalirin RNAi in combination with fyn. Scale bar=100um. (b) Scatter plots showing that fyn overexpression increases interneuronal dendritic length. The knockdown of kalirin blocked the effects of fyn on interneuron dendritic length. Knockdown of kalirin by itself did not affect dendritic length. Each point represents the total dendritic length of a single cell. Significance determined using a one-way ANOVA with Tukey post-hoc. Black lines are the mean, n=18–26 cells per condition; **p<0.01 (c) Significant interaction of fyn expression by kalirin expression with regard to total interneuron dendritic length. Significance determined using 2×2 ANOVA and the interaction term determined. Data are the mean±SEM; *p<0.05 (d) Domain mapping in hEK293 cells overexpressing various kalirin constructs in combination with fyn. Kalirin was immunoprecipitated and the immunoprecipitate probed with a phospho-tyrosine antibody. Fyn phosphorylated kalirin-7 in the presence and absence of PSD-95, and fyn phosphorylated kalirin-5. Fyn failed to phosphorylate the kalirin-7 ΔCT construct. Schematics of kalirin constructs used are shown below. (e) hEK293 cells overexpressing Y1662A or WT kalirin-7 together with PSD-95, with or without fyn. Kalirin was immunoprecipitated and the immunoprecipitate probed for phosphotyrosine. Fyn strongly phosphorylated kalirin-7. Mutation of the Y1662 kalirin-7 residue to alanine (Y1662A) significantly reduced fyn-mediated phosphorylation. Graph displays change relative to the Y1662A condition and significance determined using a one-way ANOVA with Tukey post-hoc. Data are the mean±SEM, n=4 independent experiments; *p<0.05, ***p<0.001 (f) hEK293 cells overexpressing Y1662A or WT kalirin-7 together with PSD-95, with or without fyn. Kalirin was immunoprecipitated and the immunoprecipitate probed for PSD-95. WT kalirin-7 and the Y1662A mutant showed a similar magnitude of interaction with PSD-95 in the absence of fyn. However, fyn reduced the association of WT kalirin-7, but not Y1662A kalirin-7, with PSD-95. Graph displays change relative to the Y1662A condition and significance determined using a one-way ANOVA with Tukey post-hoc. Data are the mean±SEM, n=3 independent experiments; *p<0.05

Figure 4. The Y1662 kalirin-7 residue is important for NRG1-mediated interneuronal growth

(a) hEK293 cells were transfected with PSD-95 in addition to WT kalirin-7 or Y1662A kalirin-7. ErbB4 was overexpressed in some conditions. Cells were treated with NRG1β or vehicle for 5-hours as indicated. Lysates were immunoprecipitated for kalirin-7 and subsequently probed for phospho-tyrosine. ErbB4 overexpression increased kalirin-7 tyrosine phosphorylation. Treatment of erbB4 overexpressing cells with NRG1β further increased kalirin-7 tyrosine phosphorylation, an effect not seen in cells overexpressing the Y1662A mutant. Graph displays change relative to the Kal-7+Veh. condition and significance determined using a one-way ANOVA with Tukey post-hoc. Data are the mean±SEM, n=3 independent experiments; *p<0.05, **p<0.01, ***p<0.001 (b) Representative traces of DIV28 cultured cortical multipolar interneuron dendritic trees. Axons were omitted for clarity. Neurons expressed either GFP alone or in combination with Y1662A kalirin-7 for 3-days. 1-day posttransfection, neurons were treated with either NRG1β (5nM) or with vehicle for 2 days. Scale bar=100um. (c) Overexpressed Y1662A mutant kalirin-7 is strongly targeted to interneuronal dendrites. Scale bar=35um. (d) Scatter plots. NRG1β treated interneurons overexpressing GFP show an increase in total dendritic length relative to vehicle treated cells. Inteneurons overexpressing Y1662A mutant kalirin-7 showed no significant changes in total dendritic length with either vehicle or NRG1β treatment. Each point represents the total dendritic length of a single cell. Significance determined using a one-way ANOVA with Tukey post-hoc. Black lines are the mean, n=33–37 cells per condition; *p<0.05 (e) Significant interaction of NRG1β treatment by Y1662A mutant expression with regard to total interneuronal dendritic length. Significance determined using a 2×2 ANOVA and the interaction term determined. Data are the mean±SEM; *p<0.05